key: cord-0031728-uqleqhff
authors: Song, Jia; Ge, Yuexin; Sun, Xiaoyu; Guan, Qiutong; Gong, Shiqiang; Wei, Minjie; Niu, Jumin; Zhao, Lin
title: Noncoding RNAs related to the hedgehog pathway in cancer: clinical implications and future perspectives
date: 2022-05-17
journal: Mol Cancer
DOI: 10.1186/s12943-022-01591-z
sha: 545bbfdbb7f2b82c2d4b40a10e64edad8ccc8bd7
doc_id: 31728
cord_uid: uqleqhff

Cancer is a type of malignant affliction threatening human health worldwide; however, the molecular mechanism of cancer pathogenesis remains to be elusive. The oncogenic hedgehog (Hh) pathway is a highly evolutionarily conserved signaling pathway in which the hedgehog-Patched complex is internalized to cellular lysosomes for degradation, resulting in the release of Smoothened inhibition and producing downstream intracellular signals. Noncoding RNAs (ncRNAs) with diversified regulatory functions have the potency of controlling cellular processes. Compelling evidence reveals that Hh pathway, ncRNAs, or their crosstalk play complicated roles in the initiation, metastasis, apoptosis and drug resistance of cancer, allowing ncRNAs related to the Hh pathway to serve as clinical biomarkers for targeted cancer therapy. In this review, we attempt to depict the multiple patterns of ncRNAs in the progression of malignant tumors via interactions with the Hh crucial elements in order to better understand the complex regulatory mechanism, and focus on Hh associated ncRNA therapeutics aimed at boosting their application in the clinical setting.

High mortality and recurrence rates are the major clinical hallmarks of tumors, which gravely harm human wellbeing and quality of life in the absence of definitive early symptomatic manifestation and efficient medical interventions. Constitutive activation of the hedgehog (Hh) pathway induces the expression of target genes related to cell proliferation, migration, and apoptosis, playing an indispensable role in embryogenesis and organism homeostasis [1] . Some studies have shown that the aberrant activation of the Hh pathway is linked to the formation of some cancers [2] [3] [4] . As a result, numerous inhibitors of the Hh pathway are now being investigated for therapeutic application.

Data from Human Genome Project research demonstrates that there are only less than 2% of protein-coding sequences in the human genome with the remaining 98% of the non-coding nucleic acid sequences, generally regarded as useless "garbage" and "noise", because the vast majority of them are only transcribed into RNAs, and do not continue to translate into functional proteins [5, 6] . However, the latest report launched by the Encyclopedia of DNA Elements project has shown that at least 80% of the genome, notably the remaining "junk" sequences in the human genome, is functional, with the ability to be transcribed into noncoding RNAs (ncRNAs) [7] . Employing high-throughput sequencing technology and bioinformatics methods, researchers have found that a large number of ncRNAs from extensive transcription of non-coding sequences perform important physiological and pathological functions in individuals' lives, and participate in the progress of some major disorders such as cancer, cardiovascular and neurologic diseases [8] [9] [10] [11] . At present, ncRNAs can be loosely divided into circular RNA (circRNA), microRNA (miRNA), long noncoding RNA (lncRNA), small nuclear RNA (snRNA), and so on [12, 13] by length. More and more studies prove that targeting these small molecules is a novel effective modality for cancer treatment, although ncRNAs are initially regarded as "junk" in the genome.

Despite Hh pathway inhibitors, the "off-target effects" and drug resistance hinder the radical cure of malignant tumors. Recently, the molecular mechanism of some ncRNAs regulating protein-coding genes in the tumorrelated Hh pathway has been gradually elucidated, which indicates the critical significance of ncRNAs and Hh signaling in cancer pathogenesis and provides clues for the cancer cure and recurrence elimination. This article focuses on ncRNAs, which are prominently expressed in malignancies, to study their molecular regulatory mechanisms in the Hh pathway for cancer therapy, especially circRNAs, miRNAs, lncRNAs, and snRNAs.

The Hh pathway serves a crucial purpose in early embryonic development and formation of organs and tissues, but it is dormant in adult tissues. The name "hedgehog" origins from the intrinsic mutation of the Hh gene in the cuticle of Drosophila larvae, which exhibits a spiked appearance resembling that of a frightened hedgehog [14] . In fact, Hh gene was first discovered by Nüsslein-Volhard C and Wieschaus E 40 years ago via genetic screens in Drosophila [15] . Specifically, the Hh gene family is involved in the formation of nervous system, organs, cartilages, and gonads in various vertebrates, which was reviewed by Hammerschmidt M et al. [16] . Its sequence is also isolated from invertebrates such as Hirudo medicinalis (leech), Diadema antillarum (sea urchin), and cephalochordate amphioxus (between invertebrate-vertebrate transitional stage) [17, 18] . Many human disorders, including congenital malformations, Alzheimer's disease, diabetes and malignant tumors, are undeniably linked to abnormal Hh protein activity [19] [20] [21] [22] . In addition to normal organisms, there is definite evidence that this pathway will be inappropriately activated in some human tumors, as well as related to initiation, invasion, migration, apoptotic cell death, and epithelial-mesenchymal transition (EMT) of malignant cells [1, 23] , which could be attributed to the damage repair mechanism of tumor cells.

Hh pathway signal transducers mainly consist of Hh ligand, twelve-transmembrane receptor Patched (Ptch), G protein-coupled receptor Smoothened (Smo), glioma-associated transcription factor (Gli), and other target genes. (1) Three soluble Hh ligands with lipid modification have been found in vertebrates, including sonic hedgehog (Shh), Indian hedgehog (Ihh), and desert hedgehog (Dhh). They have different distributions in different tissues, but all of them can bind to Ptch [1] . The most studied Shh is mostly distributed in the nervous system, skin and digestive tract; Ihh is primarily located in bone and cartilage; Dhh is mainly found in the gonads.

(2) In addition, the Hh protein receptor Patched with the 12-transmembrane domain has two kinds of homologs in vertebrates: Ptch1 and Ptch2, with Ptch1 in mesenchymal cells playing a leading role. (3) There are three types of transcription factors, including Gli1, Gli2, and Gli3, which play a positive or negative role at the transcriptional and post-transcriptional levels [24] . Only fulllength Gli, on the other hand, may enter the nucleus and trigger Hh target gene expression. (4) Hh potential target genes consist of hedgehog-interacting protein (Hhip) [25] , proliferative regulator N-myc proto-oncogene protein (Mycn) [26] , cell cycle regulator G1/S-specific cyclin-D1 (CCND1) [27] , D2 (CCND2) [28] , E1 (CCNE1) [29] , apoptotic regulator B-cell lymphoma 2 (Bcl2) [30] , EMT related genes snail, twist1 [31] and angiogenic regulator vascular endothelial growth factor A (VEGFA) [32] ( Table 1) .

Ptch competes with Smo for cholesterol binding due to shared sterol-binding sites, and sufficient cholesterol [43] , which has guiding significance for unraveling the intricate relationship. It's worth noting that the regulatory patterns of the Hh signaling pathway can be usually categorized as canonical and non-canonical mechanisms, the latter of which is further divided into two types: Smo-dependent and Ptch-dependent pathways, which are reviewed in detail elsewhere [2] . In addition, the above-mentioned signal transduction mechanism is referred to as the canonical Hh signaling pathway [44] (Fig. 1) .

The hallmarks of tumors are reflected in uncontrolled cell growth, gene instability, strong self-repair ability, and so on. Currently, one-third of cancers are thought to be correlated with aberrant activation of the Hh signaling pathway. Further investigations have proven that mutant or dysregulated Hh signaling could interfere with tumor behavioral phenotypes, contributing to the onset, growth, metastasis, and apoptosis of pancreatic cancer, lung cancer, ovarian cancer, breast cancer, esophageal cancer, and colorectal cancer [45] [46] [47] [48] [49] [50] . It became clear that there are three patterns of eliciting the Hh signaling cascade in multiple cancers [44, 51] (Fig. 2) :

Type 1 of Hh pathway activation is caused by activating or inactivating mutations, respectively, in the Smo gene, or the Ptch and Sufu gene, as mentioned in a previous report [52] . The study also systematically elucidated 10 skin cancer-related genes mutation in 42 patients with Ptch, and Sufu. Furthermore, the somatic mutation frequency of Ptch in studied tumors was 67% higher than previously reported. The heritable mutation of the Ptch gene in a rare autosomal dominant diseasenevoid basal cell nevus syndrome, commonly known as Gorlin Syndrome [53] was the first example of this sort of genetic mutation. Subsequently, the mutation of Ptch in sporadic medulloblastomas and other cancers was also confirmed [54] [55] [56] . In parallel, there are some reports on tumor-activator Smo and tumor-suppressor Sufu mutations in sporadic BCC, medulloblastoma and other tumors [57] [58] [59] . Thus, the core regulatory factors -Ptch, Sufu, and Smo gene mutations give us some new insights into the clinical diagnosis and treatment of human malignancies. c Type 3/a -ligand dependent oncogenic Hh pathway (paracrine). This is the paracrine mode in which stromal cells take up the Hh ligand secreted by tumor cells, and then secrete some growth factors, such as VEGF and IGF, which provide a suitable environment for tumor cells growth. d Type 3/b -ligand dependent oncogenic Hh pathway (reverse paracrine). This is the reverse paracrine mode in which tumor cells directly take up the Hh ligand secreted by stromal cells, triggering cascade response of Hh signaling

In addition to the Hh gene mutation, Hh ligand secreted by tumor cells, is also an important factor driving the Hh pathway. Hh ligand is claimed to control malignant behavior in gastric cancer [60] via autocrine regulation, including cell proliferation, which is consistent with the findings of a recent research that prove that the presence of Hh ligand is required for the occurrence of digestive tract tumors [61] . Of course, the autocrine-juxtacrine pattern is also common in a plethora of cancers including prostate cancer, multiple myeloma, gliomas, and colon cancer [62] [63] [64] [65] [66] .

The Hh ligand released by tumor cells also acts on stromal cells in another mode -the paracrine pattern. By co-culturing tumor cells and adjacent stromal cells, studies have revealed that the Hh ligand produced by tumor cells can be taken up by surrounding stromal cells which can secrete some paracrine signals (such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF) and other factors) promoting tumor growth to stimulate the Hh pathway, that is, the paracrine model [33, 67, 68] , occurred in ovarian cancer, hepatocellular carcinoma (HCC), prostate cancer, breast cancer and other cancers [3, [69] [70] [71] . Up the B-and plasma-cell malignancies lymphoma and multiple myeloma [72] , certain tumor cells directly take in Hh ligand secreted by stromal cells in the tumor microenvironment (TME) to maintain formation and survival, a process known as reverse paracrine.

Cancer stem cells (CSCs) have been known to be tumorinitiating cells. In 1997, Bonnet and his colleagues first proposed CSCs via a study on human acute myeloid leukemia [73] . The precise definition of CSCs was proposed by American Association for Cancer Research in 2006 [74] , and that is 'a cell within a tumor that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise a tumor' , which implies the necessity of targeting CSCs in tumor therapy. However, the drug resistance carried by CSCs and TME leads to malignant tumors recurrence and anticancer therapy failure.

The abnormal activity of the Hh pathway in cancer stem cell biology has been recently demonstrated in multiple myeloma [4] , colorectal cancer [75] , glioma [76] , and breast cancer [77, 78] , whereas pathway blockade lowers CSC proliferation, metastasis, and EMT. In colon cancer, Hh-Gli signaling drives stem cell survival and expansion.

In vivo experiments show that Hh-Gli pathway inhibitors cyclopamine or Smo gene silence could effectively inhibit the recurrence and metastasis of colon cancer [65] . Another study focuses on stromal cells in triple-negative breast cancer (TNBC) to see if anti-stromal therapy may be utilized to develop novel cancer treatment strategies [71] . Researchers find via using the murine M6 allograft model of TNBC that Hh ligand generated by tumor cells activates surrounding cancer-associated fibroblasts, which promotes plastic and chemotherapy-resistant phenotypes of CSCs through FGF5 activation and fibrillar collagen deposition. In the mouse model and clinical trial, Smo inhibitor treatment boosts the sensitivity of tumors to docetaxel, significantly reduces metastasis and prolongs survival. As discussed before, targeting CSCs may provide new insights into the reversal of cancer resistance via the modulation of Hh signaling.

CircRNA is a class of ncRNA molecules with no 5'cap and 3'poly A tail [79] , which is covalently closed loops with a single chain, processed by back splicing of precursor RNA [80] , with resistance to exonuclease mediated degradation, high conservation, strong stability, tissue specificity [81] [82] [83] [84] , and regulation of gene expression and malignant progression. CircRNAs can be used as miRNA or protein sponges, as well as participate in mRNA transcription and protein coding [85] . Table 2 lists recently reported circRNAs that influence Hh signaling in tumors. [43] .

Another paper [86] focuses on the association of circDGKB with the growth of neuroblastoma cells. In this research, in vitro studies clarify that overexpressed circDGKB promotes neuroblastoma cells proliferation, metastasis and tumorigenesis. This is the result of circDGKB as miR-873 sponge positive regulation of the Hh signal pathway via the upregulation of Gli1. In addition, circZNF609 [87] , as the miR-15a-5p/15b-5p sponge, promotes HCC cells proliferation, metastasis, and stemness by activating the Hh pathway through the regulation of miR-15a-5p/15b-5p and Gli2 expressions, which has been confirmed in functional tests and in vivo studies. It's reported that circZNF609 sequestering microRNA-150 plays a role in promoting colorectal cancer progression by upregulating Gli1 expression [88] . Moreover, several circRNAs such as circSMO742 [89] , circ-STAT3 [90] , circ_0036412 [91] and circDCAF6 [92] could act as miRNA sponges to affect some cancer hallmarks.

A recent investigation unveils that circGli1 interacts with p70S6K2 protein, which can block the binding of GSK3β with Gli1 and β-catenin, to facilitate Gli1 and β-catenin protein expression, activate hedgehog/ Gli1 and Wnt/β-catenin pathways and further drive Cyr61 expression by boosting their downstream gene MYC expression, leading to accelerated cell migration and angiogenesis in melanoma. These results provide strong evidence that circGli1 could be a prospective therapeutic candidate for melanoma metastasis [93] . Further investigation reveals that the highly expressed circIPO11 in HCC drives the self-renewal of liver CSCs and promotes the proliferation of HCC by recruiting TOP1 protein to the Gli1 promoter and triggering Gli1 transcription and Hh signaling pathway [94] .

MiRNA is defined as a small noncoding RNA with a length of about 22 nucleotides [95] . It mainly plays a role in regulating gene expression via participating in target mRNA degradation or translation inhibition. MicroRNA was first discovered in 1993 [96, 97] , and has gradually been widely studied in recent years. There is evidence that miRNA has been implicated in a variety of physiological and pathological processes, such as tumorigenesis, organ formation, and immune function regulation [98, 99] . Targeting miRNAs for cancer therapy has appeared to become a research hotspot in recent years, based on the relationship between abnormal activation of the Hh signaling pathway and the occurrence and development of malignant tumors. Table 3 lists recently reported miR-NAs that regulate Hh signaling in tumors. Studies have shown that most miRNAs negatively regulate the expression of target mRNA at RNA or protein levels, so miRNAs can be seen as tumor suppressor genes to participate in the cancer process. MiR205HG is identified as a novel tumor suppressor in esophageal adenocarcinoma and Barrett's esophagus [100] . The expression of miR205HG is negatively correlated with the Shh ligand of the Hh signaling pathway, according to in vivo and in vitro investigations. However, the overexpression of miR205HG effectively inhibits the expression of Ptch1. Follow-up data reveals that Ptch1 is overexpressed in Barrett's esophagus, which might be linked to its allelic mutation [129] , and further research is needed. The anti-glioma miRNA miRNA-132 [130] is considered as a research target to see how it affects the formation of gliomas. Experimental results show that upregulated miRNA-132 blocks proliferation and invasion of glioma cells by inhibiting transcriptional factor Gli1 expression [101] . [122, 123] , miR-150 [124] , miR-9 [125] , miR-224 [126] , miR-214 [127] and miR-221 [128] have been reported in tumors that induce tumor formation and recurrence through Hh signaling cascades. Of course, several miRNAs (such as miR-150) have a dual role in different tumors, which can promote or inhibit the growth of cancer. In this case, a case-by-case analysis of clinical diagnosis and treatment is required. miRNAs as downstream target genes can also be regulated by Hh signaling in addition to regulating Hh signalings. For example, a recent study shows that the transcription factor Gli2 suppresses miR-124 expression by directly binding to the upstream region of the transcriptional start site for miR-124 and AURKA is the direct target of miR-124. Overall, Gli2 drives the growth and progression of human gliomas via the miR-124/AURKA axis [117] .

LncRNA is the largest group of ncRNAs with 5′ cap and 3′ poly (A) tail and contains more than 200 nucleotides in length [131, 132] . Functional investigations reveal that lncRNAs control gene expression at epigenetic, transcriptional and post-transcriptional levels, which manipulate multiple biological processes, and are closely related to human diseases [133] [134] [135] . Indeed, lncRNAs play a regulatory role mainly by interacting with proteins or nucleotides [136] . Several lncRNAs are linked with specific signaling pathways [137] . Regardless of whether lncRNA plays a direct or indirect role in the signaling pathway, the interaction between lncRNA and signaling pathway indicates their importance in cellular processes. Here, we will focus on several regulatory mechanisms of lncRNAs at the transcriptional or post-transcriptional level. Recently reported lncRNAs that regulate Hh signaling in tumors are shown in Table 4 .

A paper reveals a new mechanism of interaction among lncRNA, miRNA and mRNA in tumorigenesis [138] . LncRNA LINC-PINT, as a tumor suppressor gene, targets miR-425-5p which targets Ptch1 of the Hh pathway to modulate laryngeal carcinoma cell stemness and chemoresistance. Several other regulatory networks comprising lncRNA, miRNA, and mRNA have been reported in the Hh-mediated cancer, including DIO3OS/miR-328/ Hhip [139] , GAS5/miR-378a-5p/Sufu [140] , LIFR-AS1/ miR-197-3p/Sufu [141] , LOC101930370/miR-1471/Shh [142] , TUG1/miR-132/Shh [143] , LINC01510/miR-335/ Shh [144] , LINC01123/miR-516b-5p/Gli1 [145] , NEAT1/ miR-503/Smo [146] and MIRLET7BHG/miR-330-5p/ Smo [147] .

Researchers identify a novel lncRNA BCAR4 that directly binds PNUTS and SNIP1 proteins, and activates transcription factor Gli2 expression in a chemokine-dependent manner [148] . In lung adenocarcinoma (LUAD), LINC01426 recruits and binds the USP22 protein to promote Shh protein stabilization, which activates the Hh pathway and promotes LUAD cells proliferation, migration, and EMT [149] . lncHDAC2 interacts with HDAC2 protein (a core component of NuRD complex [154] ) which recruits NuRD complex to the promoter of Ptch1, to resist the expression of Ptch1, thus causing abnormal activation of the Hh signaling pathway [150] . LINC01106 has been reported to facilitate proliferation and migration of colorectal cancer cells. Further investigation reveals that LINC01106 located in the nuclear could recruit FUS protein to Gli1 and Gli2 promoters, hence activating Gli1 and Gli2 transcription and promoting colorectal cancer formation [151] . Nevertheless, lncRNA HHIP-AS1 [152] acts as a tumor suppressor in HCC progression, which is attributed to its positive regulation of Hhip mRNA stability in a HuR-dependent manner.

The vast majority of cancers are closely related to the genomic changes of driver factors [155] . Although there have been a lot of investigations on cancer drivers, most of them are about coding genes, with only a handful focusing on non-coding drivers. In 2019, Suzuki H et al. [28] discovered non-coding small nuclear RNA U1-snRNA, revealing a new mechanism of abnormal splicing in cancer and providing new ideas for tumor therapy. Among snRNAs, U1 snRNA is the most abundant type of snRNAs transcribed by RNA polymerase II [156] . The study reported that in about half of Sonic hedgehog-type medulloblastomas, there is a highly recurrent hot spot mutation of A > G at the third nucleotide of snRNA, which is rarely found in other subgroups of medulloblastoma. In parallel, this mutation has a higher incidence in adults and adolescents. The U1 snRNA mutation occurs in the 5'splice site binding region, which changes the preferred base-pairing A-U to G-C, creating a new splicing junction and significantly destroying the original splicing pattern. Mechanistically, alternative splicing mediated by U1 snRNA mutations inactivates tumor suppressor gene Ptch1, promotes the expression of downstream oncogenes (Gli2, CCND2) and stimulates the Hh signaling pathway. 

Emerging evidence suggests that many ncRNAs are expected to be biomarkers for early diagnosis of cancer, reliable for predicting the prognosis of patients with cancer. Ectopic expression of ncRNAs in specific tumor tissues or circulating body fluids could be used to monitor the occurrence of neoplasms at an early stage. Previous studies show that the expression of DIO3OS is considerably downregulated in HCC tissues compared to paracancerous tissues [139] . The expression of miR-361-3p in retinoblastoma serum is also significantly downregulated [104] . The prognostic value of ncRNAs for patients with cancer is reflected in the correlation analysis between ncRNAs expression and clinicopathological features. LncRNA HOTAIR overexpression is linked to worse prognosis in patients with renal cell carcinoma [153] . In osteosarcoma, elevated levels of LINC01123 expression are related with advanced pathological staging [145] . .Remarkably, miR-324-3p expression is inversely connected with nasopharyngeal carcinoma metastasis but favorably correlated with survival time [111] . These findings suggest that Hh-related ncRNAs have broad clinical application prospects.

Therapeutic targeting of Hh signaling, as a vital cellular pathway, represents an attractive approach for the treatment of a plethora of cancers. Unremitting efforts have been made towards the development of small molecule inhibitors such as SMO antagonists-vismodegib [157] and cyclopamine [158] , Gli antagonists-GANT61 and GANT58 [159] and additional PTCH inhibitors to block Hh signaling at various sites in recent years. Yet there have been some serious drawbacks of utilizing these Hh inhibitors for the treatment of cancer, which has been discussed in detail by Javed Z et al. [160] . Hence, designing persistent small molecule inhibitors with excessive bioavailability is still a thorny question.

The multiple functional repertoires of such naturally occurring ncRNAs hold promise for the development of a potential treatment option in cancers, employing small interfering RNAs, antisense oligonucleotides together with exosomes, nanoparticles and other delivery systems [161] , which have been at the clinical trial phase. Owing to their favorable bioavailability, high specificity and good tolerance, engineered nanoformulations may be a reasonable treatment strategy for the Hh signaling mediated cancer. An in-depth analysis of the experimental data shows that PBAE/si-ciRS-7 nanocomplexes with PBAE material could significantly control the malignant behavior of renal cell carcinoma (RCC) tumors based on the carcinogenic effect of ciRS-7, providing theoretical guidance and effective basis for the treatment of RCC [162] . In line with this, researchers also construct a unique nanoparticle system to deliver anti-metastatic microRNA and conventional cytotoxic chemotherapy agents to colorectal hepatic metastases, showing miR-655-3p and oxaliplatin co-delivery system could present the inhibitory effect on tumors [163] . In addition, the successful use of a known anticancer miR-204-5p [164] [165] [166] faces many challenges including limited stability, rapid metabolism and so on. To overcome the above-mentioned drawbacks, silica nanoparticles carrying miR-204-5p and oxaliplatin are synthesized to exert their synergistic anticancer effects, leading to high apoptosis rate and limited growth of cells [167] . A novel dual delivery nanoscale device loaded with miR-345 and gemcitabine [168] is developed to treat pancreatic cancer, which greatly achieves the efficacy of miR-345. Immunohistochemical analysis from the tumor tissues of mice shows such nanocomplexes significantly downregulate Shh and its downstream effector Gli1 in the combined GEM+miR-345 treated group compared with the single gemcitabine-treated group. The encouraging findings imply that the entrance of Hh-related ncRNAs therapeutics into clinical testing is promising. Notably, nanotechnology-based system for targeted delivery of ncRNAs to the organ and cell type of interest has been applied in the field of cancer treatment, but the high cost of preparations might impede large-scale production and widespread application.

Accumulating lines of evidence have suggested that proteins and RNAs [169] can be secreted by exosomes that mediate intercellular communication, foreshadowing that exosomes can be an alternative drug delivery platform. As expected, exosomes could easily cross the biological barriers and deliver small molecules of drugs to specific tissues. For instance, exosomal TUG1 derived from cancer-associated fibroblasts promotes metastasis and glycolysis in HCC through the miR-524-5p/SIX1 axis [170] . Engineered exosomes for delivery of miR-21 and 5-fluorouracil (5-FU) are efficiently transferred into 5-FU-resistant colorectal cancer cells [171] . Combined treatment with 5-FU and miR-21 could significantly improve the anti-tumor effect of 5-FU on HCT-116 5FR cells compared with the single treatment of 5-FU or miR-21. Furthermore, co-delivery micelles system targeting miR-29b1 and GDC-0449 (also called vismodegib) might synergistically lower liver injury and improve liver fibrosis in mice [172] , which will also grant reliable insights and directions for the development of such Hh associated ncRNA therapeutics.

Accumulating evidence shows that the aberrantly activated Hh pathway confers neoplastic cells the propensity of occurrence, proliferation, and migration, which provides a new clue for researchers to explore better therapeutic strategies and drug targets for malignant tumors. Indeed, the development of drugs targeting the Hh signaling driving factors has undoubtedly opened a new door to the battle against tumors. The recognized Hh pathway inhibitors have been considered as potential therapeutic options for cancer therapy. However, owing to the presence of tumor cells heterogeneity, chemical resistance, and the complex pathway crosstalk, it is often difficult for a single pathway inhibitor, such as vismodegib, to absolutely block tumor cell proliferation. This warns us that it is urgent to design a reasonable drug delivery system and develop drugs that target other potential tumor markers.

Data from dozens of reports has indicated that noncoding RNAs can reduce the chemoresistance and stemness of tumor cells, effectively prolong the survival time of patients and reduce cancer recurrence. These small molecules usually participate in disease regulation by affecting the expression of nearby genes, especially parental genes. The ncRNAs related to the Hh pathway can directly or indirectly regulate the expression of Hh pathway genes through multifaceted molecular mechanisms and physiological processes. For example, LINC-PINT, LINC01123 and circZNF609 sponge special miRNAs to modulate target genes Ptch1, Gli1, and Gli2, respectively [87, 138, 145] . To prevent the recurrence of cancer from the perspective of eradicating tumor cells, it is quite necessary for us to consider these two thorny issues in anti-cancer treatment: (1) drug resistance, and (2) stem cells. It has been found in some cancer stem cell models that the expression of ncRNAs, such as circSmo [43] , miR-144-3p [120] , and lncHDAC2 [150] , can interfere with the stemness and chemoresistance of CSCs or tumor cells. In that respect, the appropriate combination of ncRNA-targeted agents and chemotherapeutic medicines is worthy of consideration.

The regulation mechanism of the Hh pathway is intricate and varies with the type and source of cancers. In some specific cases, several stromal cells secrete some Hh ligands or growth factors to stimulate tumor cells. The paracrine modality of the Hh pathway gives us another new anti-cancer strategy, that is, targeting normal stromal cells that shape the pre-malignant environment. It is reported that, unlike tumor cells, most stromal cells are less likely to exhibit genomic instability, which makes it difficult for them to develop Gli. The ncRNAs introduced in this review include circRNA, microRNA, lncRNA and snRNA. This diagram briefly describes the interaction between ncRNAs and five core components, with the representatives in the pentagram region associated with other Hh-related target genes resistance to therapeutic drugs. In addition, the crosstalk of Hh pathway and other pathways has been verified [173] . If diagnostic biomarkers targeting multiple pathways simultaneously can be found, it will be a big step towards curing the malignant diseases that threaten human life. As stated in the study [124] , Sufu targeted and down-regulated by oncogene miR-150 promotes the occurrence and metastasis of human gastric cancer through dual activation of Wnt/β-catenin and Hh pathways. Circular RNA circ102004 has carcinogenic effects in prostate cancer. In order to clarify the carcinogenic molecular mechanism of circ102004, western blotting assay is performed to detect the expression of key genes in the Hh pathway. It is found that circ102004 is positively correlated with the expression of P-ERK, P-AKT, P-JNK, JNK, β-catenin, and Gli1. In other words, circ102004 is involved in the regulation of some vital signaling pathways including ERK, JNK, Wnt/β-catenin and hedgehog [174] . However, the molecular pattern of circ102004 regulating various signaling pathways has not been clarified and further research remains needed.

"Off-target effects" are major barriers to the development of targeted non-coding RNA technology. The establishment of a reasonable drug delivery system can effectively reduce the failure of treatment caused by "offtarget effects". With the high-end development of pharmaceutical technology, we should consider packaging these small molecules utilizing engineered exosomes or nanoparticles to improve targeting effectiveness. If possible, the designed combination formulation of Hh inhibitors and ncRNAs based on the molecular physical and chemical properties of ncRNAs will be able to extend the time of drug action in vivo, and exert the anti-cancer effect of the medication to the greatest extent. The specific molecular mechanisms of Hh pathway need to be studied as soon as possible, which is also favorable for the successful development and design of targeted drugs. In short, this article summarizes the different roles and molecular mechanisms of ncRNAs in the Hh pathway (Fig. 3) , and sheds light on the way in which ncRNAs regulate the Hh pathway, which is conducive to diagnosis, prognostication and treatment of cancer from different angles including protein or RNA levels, and provides a new direction for the clinical therapy of tumors. 

Molecular pathways: the hedgehog signaling pathway in cancer

Targeting hedgehog signaling pathway: paving the road for cancer therapy

Hedgehog signaling promotes prostate xenograft tumor growth

Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma

International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome

Long noncoding RNAs: functional surprises from the RNA world

An integrated encyclopedia of DNA elements in the human genome

Long noncoding RNA LINC00657 inhibits cervical cancer development by sponging miR-20a-5p and targeting RUNX3

Non-coding RNAs: the key detectors and regulators in cardiovascular disease

The long noncoding RNA lncCIRBIL disrupts the nuclear translocation of Bclaf1 alleviating cardiac ischemia-reperfusion injury

The neurobiology of non-coding RNAs and Alzheimer's disease pathogenesis: pathways, mechanisms and translational opportunities

Mechanisms of endocrine resistance in breast cancer: an overview of the proposed roles of noncoding RNA

Non-coding RNA in bladder cancer

Hedgehog: functions and mechanisms

Mutations affecting segment number and polarity in drosophila

The world according to hedgehog

Products, genetic linkage and limb patterning activity of a murine hedgehog gene

The evolution of the hedgehog gene family in chordates: insights from amphioxus hedgehog

Genetics of ventral forebrain development and holoprosencephaly

Protease nexin-1 protects against Alzheimer's disease by regulating the sonic hedgehog signaling pathway

Cancer surveillance in Gorlin syndrome and Rhabdoid tumor predisposition syndrome

Elevated hedgehog-interacting protein levels in subjects with Prediabetes and type 2 diabetes

Inhibition of hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs

Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling

Vertebrate hedgehog signalling modulated by induction of a hedgehog-binding protein

A conserved HH-Gli1-Mycn network regulates heart regeneration from newt to human

LncRNA HCG18 contributes to nasopharyngeal carcinoma development by modulating miR-140/CCND1 and hedgehog signaling pathway

Recurrent noncoding U1 snRNA mutations drive cryptic splicing in SHH medulloblastoma

Hedgehog regulates cell growth and proliferation by inducing Cyclin D and Cyclin E

Activation of the BCL2 promoter in response to hedgehog/GLI signal transduction is predominantly mediated by GLI2

Twist1 and snail link hedgehog signaling to tumor-initiating cell-like properties and acquired chemoresistance independently of ABC transporters

Integrated molecular analysis of adult sonic hedgehog (SHH)-activated medulloblastomas reveals two clinically relevant tumor subsets with VEGFA as potent prognostic indicator

Hedgehog signaling in development and cancer

The mechanisms of hedgehog signalling and its roles in development and disease

Smoothened stimulation by membrane sterols drives hedgehog pathway activity

Cellular cholesterol directly activates smoothened in hedgehog signaling

Cholesterol access in cellular membranes controls hedgehog signaling

Colocalization of patched and activated sonic hedgehog to lysosomes in neurons

A mechanism for vertebrate hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes

Transducing the hedgehog signal

Activitydependent internalization of smoothened mediated by beta-arrestin 2 and GRK2

Patched1 regulates hedgehog signaling at the primary cilium

A novel protein encoded by circular SMO RNA is essential for hedgehog signaling activation and glioblastoma tumorigenicity

The role of the hedgehog signaling pathway in cancer: A comprehensive review

Sonic hedgehog paracrine signaling regulates metastasis and lymphangiogenesis in pancreatic cancer

Hedgehog signaling in lung cancer: from Oncogenesis to cancer treatment resistance

Hedgehog signaling pathway in ovarian cancer

New paradigms for the hedgehog signaling network in mammary gland development and breast cancer

Hedgehog signaling is activated in subsets of esophageal cancers

Indian hedgehog, a neglected member of hedgehog pathway, may offer a novel avenue for colorectal cancer therapy

Targeting the hedgehog pathway in cancer

Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas

Human homolog of patched, a candidate gene for the basal cell nevus syndrome

Mutations in the human homologue of the drosophila segment polarity gene patched (PTCH) in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system

Sporadic medulloblastomas contain PTCH mutations

PTCH gene mutations in invasive transitional cell carcinoma of the bladder

Activating smoothened mutations in sporadic basal-cell carcinoma

Missense mutations in SMOH in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system

Mutations in SUFU predispose to medulloblastoma

Autocrine sonic hedgehog signaling promotes gastric cancer proliferation through induction of phospholipase Cγ1 and the ERK1/2 pathway

Widespread requirement for hedgehog ligand stimulation in growth of digestive tract tumours

Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling

A critical role of autocrine sonic hedgehog signaling in human CD138+ myeloma cell survival and drug resistance

Gli activity correlates with tumor grade in plateletderived growth factor-induced gliomas

Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion

Hedgehog signalling in colon cancer and stem cells

A paracrine requirement for hedgehog signalling in cancer

Paracrine hedgehog signaling in cancer

Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis

Paracrine hedgehog signaling drives metabolic changes in hepatocellular carcinoma

Targeting stromal remodeling and cancer stem cell plasticity overcomes chemoresistance in triple negative breast cancer

Essential role of stromally induced hedgehog signaling in B-cell malignancies

Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell

Cancer stem cells--perspectives on current status and future directions: AACR workshop on cancer stem cells

RUNX3 suppresses metastasis and stemness by inhibiting hedgehog signaling in colorectal cancer

Ruiz i Altaba A. HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity

Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells

LncRNA-Hh strengthen cancer stem cells generation in twist-positive breast cancer via activation of hedgehog signaling pathway

Detecting and characterizing circular RNAs

Regulation of circRNA biogenesis

Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing

Circular RNAs are abundant, conserved, and associated with ALU repeats

Circular RNAs are a large class of animal RNAs with regulatory potency

Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals

The biogenesis, biology and characterization of circular RNAs

Circular RNA DGKB promotes the progression of neuroblastoma by targeting miR-873/GLI1 Axis

CircZNF609 enhances hepatocellular carcinoma cell proliferation, metastasis, and stemness by activating the hedgehog pathway through the regulation of miR-15a-5p/15b-5p and GLI2 expressions

Circ-ZNF609 promotes migration of colorectal cancer by inhibiting Gli1 expression via micro-RNA-150

Circular RNA SMO sponges miR-338-3p to promote the growth of glioma by enhancing the expression of SMO

Transcription activation of circ-STAT3 induced by Gli2 promotes the progression of hepatoblastoma via acting as a sponge for miR-29a/b/c-3p to upregulate STAT3/Gli2

Circ_0036412 affects the proliferation and cell cycle of hepatocellular carcinoma via hedgehog signaling pathway

Circ_DCAF6 potentiates cell stemness and growth in breast cancer through GLI1-hedgehog pathway

Circ-GLI1 promotes metastasis in melanoma through interacting with p70S6K2 to activate hedgehog/GLI1 and Wnt/β-catenin pathways and upregulate Cyr61

Circular RNA circIPO11 drives self-renewal of liver cancer initiating cells via hedgehog signaling

Approaches to microRNA discovery

The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14

Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans

MicroRNA therapeutics: towards a new era for the management of cancer and other diseases

Micro-RNAs in the regulation of immune response against SARS CoV-2 and other viral infections

Novel Long noncoding RNA miR205HG functions as an esophageal tumorsuppressive hedgehog inhibitor

miR-132 weakens proliferation and invasion of glioma cells via the inhibition of Gli1

Targeting the SMO oncogene by miR-326 inhibits glioma biological behaviors and stemness

Targeting of the signal transducer Smo links micro-RNA-326 to the oncogenic hedgehog pathway in CD34+ CML stem/ progenitor cells

MicroRNA-361-3p regulates retinoblastoma cell proliferation and stemness by targeting hedgehog signaling

miRNA-338-3p suppresses cell growth of human colorectal carcinoma by targeting smoothened

miR-338-3p suppresses invasion of liver cancer cell by targeting smoothened

MicroRNA-182-5p regulates hedgehog signaling pathway and chemosensitivity of cisplatin-resistant lung adenocarcinoma cells via targeting GLI2

MicroRNA-1271 inhibits proliferation and promotes apoptosis of multiple myeloma cells through inhibiting smoothened-mediated hedgehog signaling pathway

Downregulation of miR-506-3p facilitates EGFR-TKI resistance through induction of sonic hedgehog signaling in non-small-cell lung cancer cell lines

MiR-873-5P controls gastric cancer progression by targeting hedgehog-GLI signaling

The expression of microRNA-324-3p as a tumor suppressor in nasopharyngeal carcinoma and its clinical significance

MiR-218 inhibits multidrug resistance (MDR) of gastric cancer cells by targeting hedgehog/ smoothened

miR-636 inhibits EMT, cell proliferation and cell cycle of ovarian cancer by directly targeting transcription factor Gli2 involved in hedgehog pathway

miR-506 acts as a tumor suppressor by directly targeting the hedgehog pathway transcription factor Gli3 in human cervical cancer

miR-7-5p acts as a tumor suppressor in bladder cancer by regulating the hedgehog pathway factor Gli3

DNA-methylationmediated silencing of miR-7-5p promotes gastric cancer stem cell invasion via increasing Smo and Hes1

Inhibition of the hedgehog signaling pathway suppresses cell proliferation by regulating the Gli2/miR-124/AURKA axis in human glioma cells

Targeting of miR-150 on Gli1 gene to inhibit proliferation and cell cycle of esophageal carcinoma EC9706

CLL Exosomes modulate the Transcriptome and behaviour of recipient stromal cells and are selectively enriched in miR-202-3p

MiR-144-3p inhibits gastric cancer progression and stemness via directly targeting GLI2 involved in hedgehog pathway

miR-141-3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells

miR-212 promotes pancreatic cancer cell growth and invasion by targeting the hedgehog signaling pathway receptor patched-1

MicroRNA-212 displays tumor-promoting properties in non-small cell lung cancer cells and targets the hedgehog pathway receptor PTCH1

Dual activation of hedgehog and Wnt/β-catenin signaling pathway caused by downregulation of SUFU targeted by miRNA-150 in human gastric cancer

Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level

Down-regulation of microRNA-224 -inhibites growth and epithelial-to-mesenchymal transition phenotype -via modulating SUFU expression in bladder cancer cells

microRNA-214 promotes epithelial-mesenchymal transition and metastasis in lung adenocarcinoma by targeting the suppressor-of-fused protein

MicroRNA-221 promotes tumor progression by targeting HHIP in human glioblastoma

Genomic landscape of somatic alterations in esophageal squamous cell carcinoma and gastric cancer

HLF/miR-132/TTK axis regulates cell proliferation, metastasis and radiosensitivity of glioma cells

The novel regulatory role of lncRNA-miRNA-mRNA axis in cardiovascular diseases

The how and why of lncRNA function: An innate immune perspective

The long noncoding RNA Chaer defines an epigenetic checkpoint in cardiac hypertrophy

Local regulation of gene expression by lncRNA promoters, transcription and splicing

Transcriptional and post-transcriptional gene regulation by Long non-coding RNA

Gene regulation by long non-coding RNAs and its biological functions

LncRNA-mediated regulation of cell signaling in cancer

Long noncoding RNA LINC-PINT regulates laryngeal carcinoma cell stemness and chemoresistance through miR-425-5p/PTCH1/SHH axis

Long noncoding RNA DIO3OS hinders cell malignant behaviors of hepatocellular carcinoma cells through the microRNA-328/Hhip Axis

lncRNA GAS5-promoted apoptosis in triple-negative breast cancer by targeting miR-378a-5p/SUFU signaling

LIFR-AS1 modulates Sufu to inhibit cell proliferation and migration by miR-197-3p in breast cancer

research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year submit your research ? Choose BMC

The long noncoding RNA TUG1 acts as a competing endogenous RNA to regulate the hedgehog pathway by targeting miR-132 in hepatocellular carcinoma

SOX2-induced upregulation of lncRNA LINC01510 promotes papillary thyroid carcinoma progression by modulating miR-335/SHH and activating hedgehog pathway

LINC01123 enhances osteosarcoma cell growth by activating the hedgehog pathway via the miR-516b-5p/Gli1 axis

Silencing of long non-coding RNA NEAT1 inhibits hepatocellular carcinoma progression by downregulating SMO by sponging microRNA-503

MIRLET7BHG promotes hepatocellular carcinoma progression by activating hepatic stellate cells through exosomal SMO to trigger hedgehog pathway

lncRNA directs cooperative epigenetic regulation downstream of chemokine signals

Upregulation of LINC01426 promotes the progression and stemness in lung adenocarcinoma by enhancing the level of SHH protein to activate the hedgehog pathway

The long non-coding RNA LncHDAC2 drives the self-renewal of liver cancer stem cells via activation of hedgehog signaling

LINC01106 drives colorectal cancer growth and stemness through a positive feedback loop to regulate the Gli family factors

A novel long noncoding RNA HHIP-AS1 suppresses hepatocellular carcinoma progression through stabilizing HHIP mRNA

HOTAIR and androgen receptor synergistically increase GLI2 transcription to promote tumor angiogenesis and cancer stemness in renal cell carcinoma

Cancer biology and NuRD: a multifaceted chromatin remodelling complex

A compendium of mutational cancer driver genes

Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells

Efficacy and safety of vismodegib in advanced basal-cell carcinoma

Effects of oncogenic mutations in smoothened and patched can be reversed by cyclopamine

Inhibition of GLImediated transcription and tumor cell growth by small-molecule antagonists

Regulation of hedgehog signaling by miRNAs and Nanoformulations: A possible therapeutic solution for colorectal cancer

Noncoding RNA therapeutics -challenges and potential solutions

ciRS-7 is a prognostic biomarker and potential gene therapy target for renal cell carcinoma

In vivo delivery and therapeutic effects of a MicroRNA on colorectal liver metastases

miR-204-5p inhibits proliferation and invasion and enhances chemotherapeutic sensitivity of colorectal cancer cells by downregulating RAB22A

Tumor suppressor miRNA-204-5p regulates growth, metastasis, and immune microenvironment remodeling in breast cancer

MicroRNA-204-5p is a tumor suppressor and potential therapeutic target in head and neck squamous cell carcinoma

MiRNA-204-5p and oxaliplatin-loaded silica nanoparticles for enhanced tumor suppression effect in CD44-overexpressed colon adenocarcinoma

Dual delivery nanoscale device for miR-345 and gemcitabine co-delivery to treat pancreatic cancer

RNA delivery by extracellular vesicles in mammalian cells and its applications

Exosomal lncRNA TUG1 from cancer-associated fibroblasts promotes liver cancer cell migration, invasion, and glycolysis by regulating the miR-524-5p/SIX1 axis

Engineered exosomes for targeted co-delivery of miR-21 inhibitor and chemotherapeutics to reverse drug resistance in colon cancer

Co-delivery of small molecule hedgehog inhibitor and miRNA for treating liver fibrosis

Opposite regulation of Wnt/β-catenin and Shh signaling pathways by Rack1 controls mammalian cerebellar development

Upregulated circular RNA circ-102004 that promotes cell proliferation in prostate cancer

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations